Genome Wide Association Studies Using a New

Nonparametric Model Reveal the Genetic Architecture of

17 Agronomic Traits in an Enlarged Maize Association
Panel
Ning Yang1., Yanli Lu1,2., Xiaohong Yang3, Juan Huang1, Yang Zhou1, Farhan Ali1, Weiwei Wen1,
Jie Liu1, Jiansheng Li3, Jianbing Yan1*
1 National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, China, 2 Maize Research Institute, Sichuan Agricultural University,
Wenjiang, Sichuan, China, 3 National Maize Improvement Center of China, Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Beijing,
China

Abstract
Association mapping is a powerful approach for dissecting the genetic architecture of complex quantitative traits using
high-density SNP markers in maize. Here, we expanded our association panel size from 368 to 513 inbred lines with 0.5
million high quality SNPs using a two-step data-imputation method which combines identity by descent (IBD) based
projection and k-nearest neighbor (KNN) algorithm. Genome-wide association studies (GWAS) were carried out for 17
agronomic traits with a panel of 513 inbred lines applying both mixed linear model (MLM) and a new method, the
Anderson-Darling (A-D) test. Ten loci for five traits were identified using the MLM method at the Bonferroni-corrected
threshold 2log10 (P) .5.74 (a = 1). Many loci ranging from one to 34 loci (107 loci for plant height) were identified for 17
traits using the A-D test at the Bonferroni-corrected threshold 2log10 (P) .7.05 (a = 0.05) using 556809 SNPs. Many known
loci and new candidate loci were only observed by the A-D test, a few of which were also detected in independent linkage
analysis. This study indicates that combining IBD based projection and KNN algorithm is an efficient imputation method for
inferring large missing genotype segments. In addition, we showed that the A-D test is a useful complement for GWAS
analysis of complex quantitative traits. Especially for traits with abnormal phenotype distribution, controlled by moderate
effect loci or rare variations, the A-D test balances false positives and statistical power. The candidate SNPs and associated
genes also provide a rich resource for maize genetics and breeding.

Citation: Yang N, Lu Y, Yang X, Huang J, Zhou Y, et al. (2014) Genome Wide Association Studies Using a New Nonparametric Model Reveal the Genetic
Architecture of 17 Agronomic Traits in an Enlarged Maize Association Panel. PLoS Genet 10(9): e1004573. doi:10.1371/journal.pgen.1004573
Editor: Rodney Mauricio, University of Georgia, United States of America
Received September 19, 2013; Accepted June 30, 2014; Published September 11, 2014
Copyright: ß 2014 Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by the National Natural Science Foundation of China (31123009, 31161140347, 31222041: http://www.nsfc.gov.cn/Portal0/
default152.htm) and the National Hi-Tech Research and Development Program of China (2012AA10A307: http://www.863.gov.cn/). YL was partly supported by
the open funds of the National Key Laboratory of Crop Genetic Improvement. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: yjianbing@mail.hzau.edu.cn
. These authors contributed equally to this work.

Introduction
Maize (Zea mays L.) is one of the most important food, feed and
industrial crops globally. Grown extensively under different
climate conditions across the world, maize shows an astonishing
amount of phenotypic diversity [1]. Identifying the underlying
natural allelic variations for the phenotypic diversity will have
immense practical implications in maize molecular breeding for
improving nutritional quality, yield potential, and stress tolerance.
With the rapid development of next generation sequencing and
high-density marker genotyping techniques, there emerges tre-
mendous interest in using association mapping to identify genes
responsible for quantitative variation of complex traits [2]. The use
of GWAS has been well demonstrated in model plants such as
Arabidopsis [3] and rice [4]. In maize, we examined the genetic
architecture of maize oil biosynthesis in 368 diverse maize inbred
lines with over 1.06 million SNPs obtained from RNA sequencing
and DNA array using the GWAS strategy [5]. Despite the great
potential that GWAS has to pinpoint genetic polymorphisms
underlying agriculturally important traits, false discoveries are a
major concern and can be partially attributed to spurious
associations caused by population structure and unequal related-
ness among individuals in a given panel [6]. A number of statistical
approaches have been proposed, among which the mixed linear
model (MLM) is one of the popular methods that can eliminate the
excess of low p values for most traits [6,7]. However, Zhao et al.
[8] performed GWAS using a NAÏVE model in each sub-
population and MLM with inferred population structure as a fixed
effect in the whole mapping panel of rice, and their results
suggested that MLM may lead to false negatives by overcompen-
sating for population structure and relatedness. To improve the
MLM, some strategies to best utilize marker data have been

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Author Summary
Genotype imputation has been used widely in the analysis
of genome-wide association studies (GWAS) to boost
power and fine-map associations. We developed a two-
step data imputation method to meet the challenge of
large proportion missing genotypes. GWAS have uncov-
ered an extensive genetic architecture of complex quan-
titative traits using high-density SNP markers in maize in
the past few years. Here, GWAS were carried out for 17
agronomic traits with a panel of 513 inbred lines applying
both mixed linear model and a new method, the
Anderson-Darling (A-D) test. We intend to show that the
A-D test is a complement to current GWAS methods,
especially for complex quantitative traits controlled by
moderate effect loci or rare variations and with abnormal
phenotype distribution. In addition, the traits associated
QTL identified here provide a rich resource for maize
genetics and breeding.
proposed [9,10]. The more we know about the genetics of a trait,
the greater our power is to detect the rest of the genetic
contribution. The problem is, of course, that we usually do not
know what the causal loci are, and methods that try to identify
them are prone to over-fitting [11]. Beló et al. [12] adopted the
Kolmogorov–Smirnov (KS) test for association analysis in each
subpopulation of the mapping panel and an allelic variant of fad2
associated with increased oleic acid level was successfully identified
based on modest density markers. However, detailed instructions
for the algorithm were not published. Most current GWAS
methods lack the power to detect rare alleles and this has limited
the application of GWAS, since rare alleles are common in maize
diversity collections [1,5]. Parametric tests of association are
sensitive to SNPs with minor allele frequencies, which can
artificially increase association scores. Balancing samples across
population subdivisions can homogenize allele frequencies,
elevating rates of globally rare variants that are common in
certain subdivisions [5].
In this study, 513 diverse maize inbred lines [13], representing
tropical/subtropical and temperate germplasm, were genotyped
by MaizeSNP50 BeadChip containing 56,110 SNPs [14]. RNA
sequencing (RNA-seq) was performed on 368 of these 513 lines
and 556,809 high quality SNPs with a minor allelic frequency
greater than 0.05 were obtained [5,15,16]. Seventeen agronomic
traits were systematically phenotyped for the 513 lines under
multiple environments and seasons (see Materials and Methods).
The objectives of this research were (1) to explore an efficient
imputation method to infer missing genotypes for the 145 inbreds
that were only genotyped by SNP-chip (low density), not by RNA-
seq (high density); (2) to develop a powerful statistical method for
GWAS to identify robust QTL for complex agronomic traits in
maize; and (3) to methodically analyze the underlying genetic
architectures of the 17 agronomic traits in the diverse maize
association mapping panel.
Results
Phenotypic variation for 17 agronomic traits
A brief description of each trait, its acronym, and evaluation
methodology was summarized in Supplementary Table S1. All of
the 17 traits in the 513 maize inbred lines were in accordance with
a normal distribution (Figure S1A, S2, S3, S4, S5, S6, S7, S8, S9,
S10, S11, S12, S13, S14, S15, S16, S17A). But the phenotype of
each trait showed distinct differences among four subgroups
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Genome Wide Association Studies in Maize
(Figure S1B, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13,
S14, S15, S16, S17B). Analysis of Variance (ANOVA) showed that
population structure explained 39.5% of phenotypic variation
(PVE) for tassel main axis length, which was the highest among the
17 agronomic traits included in best linear unbiased prediction
(Table S2), indicating vulnerability of this particular trait to the
population structure and variable sensitivity of different traits to
population structure. Furthermore, heritability (h2) was highest
(0.683) for tassel main axis length (TMAL), while the lowest
heritability (0.386) was observed for kernel number per row
(KNPR) among the traits. Pair-wise Pearson’s correlation coeffi-
cients of the 17 traits revealed that phenotypes within a category
were more correlated. The values ranged from 0.001 between
kernel width and plant height to 0.95 between days to anthesis and
heading (Figure S18). These results indicated that all the tested
lines possessed significant genetic variability and can be used for
further genetic analyses.
SNP projection and imputation
The whole panel, 513 maize inbred lines, was genotyped using
the MaizeSNP50 BeadChip containing 56,110 SNPs (Illumina).
RNA sequencing was performed on immature seeds for 368 out of
the 513 maize inbreds using 90-bp paired-end Illumina sequenc-
ing, resulting in 2,445.9 Gb of raw sequencing data. 556,809 high
quality SNPs obtained by combining the two genotyping platforms
(RNA-seq and SNP array) [5,16] were used in the study. For the
additional 145 maize lines, the genotype calls of unique loci from
the integrated SNP data were projected based on regions of IBD to
physical maps constructed using 56110 SNPs, and then high-
density markers with more than 0.5 million SNPs were obtained
for all the lines. Out of 56,110 SNPs from MaizeSNP50 data set,
49728 SNPs overlapped with the integrated SNPs data based on
their physical positions (B73 RefGen_v2). The 49,728 common
SNPs were regarded as core or frame markers for projection based
on IBD regions. In order to evaluate the performance of IBD [17]
based projection, training and validation datasets were established
for chromosome 1, which had 7818 core markers from Illumina
Maize SNP50 and 88581 SNPs from the integrated data set. The
genotypes for one maize line with RNA-seq data in IBD regions
were assigned to the matched target line without RNA-seq data for
each SNP. The projection accuracy was calculated by comparing
inferred genotypes of 368 lines with their real genotype obtained
from RNA-seq. In addition, KNN algorithm [4] which infers a
large number of missing genotypes generated from low-coverage
genome sequencing was used to impute the missing genotypes of
the unique loci from RNA-seq SNP data based on 49728 frame
markers. Single method analysis, either IBD based projection or
KNN algorithm, cannot achieve both optimal accuracy and
coverage (see Materials and Methods). However, the combination
of IBD based projection and KNN seemed effective to infer a large
number of missing genotypes. In order to optimize the set of
imputation parameters, a simulation was performed on chromo-
some 1 in 368 lines (Figure S19). The simulation result on
chromosome 1 in 368 lines indicated that the missing rate was
reduced from 91.6% (1–7,818/88,581) to 12.8%, with an accuracy
rate 96.6% (Table S3). The optimal parameter combination (IBD:
SNPs number$150 in 5 Mb window size; KNN: w = 20, k = 6,
p = 27, r = 1) was then used to impute the missing SNPs for the
remaining 145 inbred lines, resulting in an 85.5% filling rate.
Therefore, our approach combining SNP-chip data and RNA-seq
SNP data with an effective projection procedure permits the quick
construction of a high-density physical map and integration of
SNPs from RNA-seq data set onto the whole population. This
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approach is also applicable to other genomes and genotyping data
from different platforms for a variety of downstream analyses.
Statistical power of the imputation-based association
test
The 368 maize inbreds with 556,809 SNPs genotyped by RNA-
seq and Maize SNP50 array were defined as Data set 1. In
addition, Data set 1 and 145 maize inbred lines with joint IBD-
based projection and KNN imputed genotypes were defined as
Data set 2 together. The 513 maize inbreds with Maize SNP50
array genotyped were defined as Data set 3. To evaluate the
reliability of imputed genotypes for 145 inbred lines, GWAS was
performed using MLM, with both Data sets 1, 2 and 3 focusing on
kernel oil concentration, which has been thoroughly analyzed in
our previous study [5].
For GWAS performed using MLM with both Data sets 1 to 3, a
total of 26, 32 and 8 significant loci were identified in Data sets 1
to 3, respectively, at the Bonferroni-corrected threshold (2log P.
5.74, a = 1,) (Figure 1). Almost all strong signals identified in data
sets 1 and 3 were also identified in data set 2 (Table S4). More
interestingly, we identified six additional significantly associated
loci in dataset 2 (2log P.5.74, a = 1), including the phosphoino-
sitide 3-kinases gene (PI3Ks) and the phosphatidylinositol transfer
protein, which is known to be involved in the oil concentration
trait [18] (Figure 1, Table 1, Table S4). This suggests that GWAS
carried out using the imputed genotypes with a larger population
(n = 513) increased the statistical power compared to the analyses
of RNA-seq genotyped SNPs with the smaller population size
(n = 368) or low density as DNA array SNPs with the same
population size (n = 513).
GWAS for 17 agronomic traits using MLM
GWAS for 17 agronomic traits using MLM was conducted with
Data set 2 and the results are summarized in Figures S1C, D, S2,
S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16,
S17C, D. A total of 19 significant SNPs from 10 loci were
identified for five traits (ear leaf width, ear length, kernel width,
plant height and tassel main axis length) (Table 2). No significant
SNP was found to be associated with the other 12 tested traits at
the Bonferroni-corrected threshold (2log P.5.74, a = 1). If we set
Figure 1. Comparison of mapping results for kernel oil concentration in two different data sets. (A) Manhattan plots of mixed linear
model conducted in data set 1 and 2, respectively (data set 1: n = 368, without imputed genotypic data; data set 2: n = 513, 145 lines with imputed
genotypic data). The arrow and red boxes indicate the new loci that were not identified in previous study (Li et al, 2013); (B) Quantile-Quantile plots of
p-values of mixed linear model conducted in data sets 1 and 2, respectively.
doi:10.1371/journal.pgen.1004573.g001
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Genome Wide Association Studies in Maize
the Bonferrroni-corrected threshold as 2log P.7.05 (a = 0.05), no
SNPs were significant for all the 17 traits. It may be too strict to
use 0.05/n as the cutoff since not all the markers are independent.
One thousand permutation tests were conducted for three typical
traits with different level of population structure (kernel width, ear
height and days to heading) (Table S2). The results showed the
cutoff value at a = 0.05 is quite similar (Table S5) with 0.05/n.
Anderson-Darling (A-D) test, an alternative method for
GWAS
The A-D test [19] is a nonparametric statistical method and a
modification of the KS test [12,20] that gives more weight to the
tails of the distribution than the KS test. Since the identified loci
were much less numerous than expected using the MLM method,
the data set was reanalyzed using the A-D test. The same three
traits (kernel width, ear height and days to heading) were used to
perform 1000 time permutation tests to determine the cutoff
values. The results showed the cut off value at a = 0.05 varied
around the Bonferrroni-corrected threshold as 0.05/n (Table S5).
To simplify the procedures, we used the uniform cutoff (2log P.
7.05, a = 0.05) for further analysis. Flowering time is an important
and well-studied trait, and many QTL or candidate genes have
been identified [21,22]. Recently, several studies have confirmed
that ZmCCT is the gene underlying the major QTL affecting
flowering time on chromosome 10 [22,23]. Taking flowering time
as an example, it provides a good opportunity to test whether A-D
test is a feasible GWAS method for agronomic traits or not. Using
the A-D test, we identified 30 loci associated with days to heading
in Yunnan 2010. Around 20% of 30 loci were located within a
QTL support interval reported in NAM population [22]. If the
significant loci are randomly distributed in the genome, the
probability by chance is equal to the ratio between the whole-
length of QTL interval and the whole genome length (12%), which
represents an almost twofold enrichment compared with the 12%
expected by chance. A strong association (2log10 (P) = 7.59) was
identified in 1.7 Kb upstream of ZmCCT (Figure 2A). Four other
loci seem to be strong candidates including: one homologous gene
(CIB1) [24] shown to be involved in the regulation of flowering
time in Arabidopsis, two homologues containing CCT domain that
was demonstrated as key photoperiod regulatory gene in plants
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 Table 1. Additional SNPs and candidate genes significantly associated with oil concentration found using imputed genotype data.

Candidate gene Chr Position SNP *Alleles MAF eQTL P(n = 368) P(n = 513) Annotation

GRMZM2G132898 1 105672248 M1c105672248 G/T 0.07 N.S. 9.21E-05 1.58E-06 choline-phosphate

cytidylyltransferase,CCT
GRMZM2G355572 3 7938035 M3c7938035 A/C 0.09 N.S. 5.39E-06 1.25E-06 Unknown
GRMZM2G142315 3 156963535 M3c156963535 C/G 0.05 6.70E-12 5.42E-06 8.46E-08 phosphatidylinositol transfer
protein,CSR1
GRMZM2G122277 4 31245730 PZE-104026172 A/C 0.11 N.S. 1.91E-049 7.87E-07 Unknown
GRMZM2G138245 5 22806147 M5c22806147 A/G 0.06 8.59E-13 4.77E-05 2.14E-07 Phosphoinositide 3-kinase,PI3Ks

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GRMZM2G467356 7 139139746 M7c139139746 C/G 0.06 N.S. 2.93E-05 4.75E-08 Unknown

*Alleles with underlines indicate rare alleles.

N.S: non-significant.
doi:10.1371/journal.pgen.1004573.t001

4
Table 2. Comparison of significant SNPs identified for 17 traits from MLM using imputed data (dataset 2).

Traits Chr. Position SNPs Candidate genes Annotation 2Log10 (P)* PVE (%)

EL 1 50646115 A/G GRMZM2G329040 Haloacid dehalogenase-like hydrolase 6.91 5.32

1 50668188 A/C GRMZM2G703565 Thioredoxin-like fold 6.82 5.41
1 50676605 A/C GRMZM2G008490 Unknown 6.82 5.2
1 50712263 T/C AC208571.4_FG001/GRMZM5G851485 Six-bladed beta-propeller, TolB-like/Helix-loop-helix domain 6.71 3.5
TMAL 1 229858329 T/C GRMZM2G079428 Unknown 5.76 5.52
1 285931176 A/G GRMZM2G434533 Protein kinase, catalytic domain 6.18 5.17
PH 3 162709488 A/G GRMZM2G401050 Unknown 6.56 9.61
KW 7 148464475 A/C GRMZM2G413044 Unknown 5.83 0.03
EL 8 101507590 A/G GRMZM2G164090 Gibberellin regulated protein 6.05 4.23
ELW 10 126586283 A/G GRMZM2G167280 Protein kinase, catalytic domain 5.81 5.73

*The Bonferroni –corrected threshold is 2log10 (P).5.74 (the Bonferroni-corrected thresholds for the P values were 1.79661026 and corresponding 2log10 (P) values of 5.74 for 556809 SNPs, at a = 1).
Chr, chromosome; PVE, explained phenotypic variation; EL, ear length; DTH, days to heading; TMAL, tassel main axis length; PH, plant height; ED, ear diameter; KW, kernel width; DTA, days to anthesis; ELW, ear leaf width.
doi:10.1371/journal.pgen.1004573.t002
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[25], and one locus previously shown to affect flowering time in
maize (Id1) [26] (Figure 2A). Using the MLM method, we were
only able to identify the marginally significant association for
ZmCCT (2log10 (P) = 5.64) and there were no strong signals in
other genome regions (Figure 2B). Therefore, A-D test could be a
more appropriate GWAS method for agronomic traits and we
performed GWAS using the A-D test in each subpopulation of
Data set 2 without controlling of population structure for all tested
traits. The total number of unique SNPs significantly associated
with the 17 traits was 678, of which 310 represented unique loci
(Figure S1E, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13,
S14, S15, S16, S17E). The numbers of significant SNPs associated
with different traits ranged from 1 (Cob Weight) to 35 (Tassel
branch number and Days to silking). For plant height, a total of
107 loci were identified at the Bonferroni-corrected threshold (2
log P.7.05, a = 0.05) (Table S6, S7). About 10% of the loci were
detected to affect two or more different traits that were consistent
with the observed correlations among the measured traits (Figure
S18). There were 101, 71 and 171 loci detected in three
subpopulations: SS (subpop-1), NSS (subpop-2) and TST (sub-
pop-3), respectively. A reasonable number of spurious associations
should be existed in the detected loci since the population structure
is not properly addressed in each subpopulation. Genomic control
[27–30] is a popular method to control the population stratifica-
tion and cryptic relatedness that was applied to adjust A-D test
statistic in each subpopulation in present study. In total, 19 loci
were significantly associated with 13 traits at the Bonferroni-
corrected threshold (2log P.5.74, a = 1) (Table S7).
To further examine the nature of statistically significant
associations, we examined the phenotype distributions of individ-
uals carrying each allele. Interestingly, some associations that
differed in the width of the phenotype distribution but which had
nearly identical trait means were found to be highly significant by
the A-D test but not significant by MLM. Figure 3 illustrated
Figure 2. GWAS of the phenotype of days to heading in Yunnan 2010. (A) GWAS result by Anderson–Darling test; (B) GWAS result by mixed
linear model.
doi:10.1371/journal.pgen.1004573.g002
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Genome Wide Association Studies in Maize
significant loci for ear height with nearly identical trait means
(Figure 3A) and significant loci for ear leaf width with an obvious
shift of the means (Figure 3B). In total, 14.6% of significant loci
identified by A-D test do not have an obvious shift of the mean
between the two alleles (t-test, p.0.05) (Table S7). In this case the
differences between distributions are real, but the corresponding
genetic markers would not be useful in breeding if the objective is
to change the phenotypic means.
Comparison of different association mapping methods
based on simulated data
Causal allele frequencies and trait distributions are the main
factors that affect association mapping efficiency [1,31]. GWAS
data were simulated by adding phenotypic effects to real genotypic
data considering the population structure and epistasis from
MaizeSNP50 BeadChip [13] under three scenarios: a normal
distribution model, an abnormal distribution model caused by
uncertain effectors like phenotyping errors and an abnormal
distribution model caused by a larger effect QTN with rare alleles
(Methods). We compared our noticed A-D test with three other
mapping methods: Kruskal-Wallis (K-W) test and linear model
(LM) which does not correct for population structure; MLM which
corrects for population structure and kinship. Statistic power of the
four methods were compared under the same level Type I error.
For each method, QTNs were considered to be detected if their P
value were below the threshold determined by 1,000 times
permutation.
The results for the three simulation schemes are shown in
Figure 4 and can be summarized as follows: First, MLM has more
(Figure 4A, C, I) or similar (Figure 4G) power among the four
methods for major QTNs in schemes 1 and 3. Second, regardless
of the allele frequency, nonparametric methods usually have
greater power than LM and MLM for moderate QTNs
(Figure 4D–F, J–I). Third, A-D test is more powerful than K-W
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Figure 3. The nature of statistically significant associations. The illustration of associations those are highly significant by Anderson–Darling
test, with nearly identical trait means for ear height (A) or with an obvious shift of the means for ear leaf width (B).
doi:10.1371/journal.pgen.1004573.g003
test in terms of QTNs with rare alleles (Figure 4J–I). Fourth,
nonparametric methods are much more powerful than parametric
methods in scheme 2 (Figure 4B, E, H, K) that the phenotype has
an abnormal distribution model caused by uncertain effectors.
Co-localization of QTL and candidate genes for
agronomic traits
We compared our mapping results for 17 agronomic traits with
QTL identified using different linkage segregation populations and
with previously reported known genes. Loci identified by the A-D
test which overlap with previously identified genes and loci
mapped in biparental populations are summarized in Table S7.
Considering the large confidence interval of previously reported
QTL, 3 independent RIL populations genotyped with high-
density SNP markers were used to conduct QTL analysis for 3
traits (kernel width, ear length and kernel number per row) of the
17 traits tested. For the compared traits, 9 loci (20% of the
detected loci) identified by the A-D test were within the QTL
confidence interval. One example is a major kernel width QTL
which was mapped in chromosome 7 with BK/Yu8701 RILs and
explains 18.7% of phenotypic variation (Figure 5B). Within the
QTL interval, significant SNPs- kernel width association was
detected (Figure 5A). Six candidate genes: GRMZM2G354539,
GRMZM2G052893, GRMZM2G052817, GRMZM2G354525,
GRMZM2G052610, and GRMZM2G052509 are found in the
associated interval. An expression quantitative trait locus (eQTL)
was detected for one (GRMZM2G052509, 2log10 (P) = 10.16) of
the six annotated genes, which can therefore be regarded as a
candidate gene for further study. The second example of overlap
included the SNP chr2.s_1972207(C/G) with 2log10 (P) = 9.06and
SNP chr2.s_1972176(C/G) with 2log10 (P) = 8.49 which were both
significantly associated with ear length, and a QTL affiliated with
ear length identified in B73/By804 RIL near the associated peak
(Figure 5D–F). SNP chr2.s_1972207 (C/G) and SNP chr2.s_1972176
(C/G) were the only two of the 36 SNPs within the gene
GRMZM2G061877, which encodes a DHHC zinc finger domain
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Genome Wide Association Studies in Maize
containing protein, and both of them are in the CDS region. SNP
chr2.s_1972176 (C/G) makes no difference to the translated protein
sequence, while SNP chr2.s_1972207 (C/G) results in a change of
Isoleucine to Methionine. Several zinc finger proteins that play
important roles in maize inflorescence development, for instance
transcription factors RA1, RA2 and RA3 in ramosa pathways [32],
have been identified. In rice, a zinc finger transcription factor DST
directly regulates OsCKX2 expression in the reproductive meristem
leading to OsCKX2 regulated CK accumulation in the shoot apical
meristem (SAM) and, therefore, controls the number of the
reproductive organs; the dst mutant leads to lower plant height
and longer rice panicle length [33]. These zinc finger genes are
functioning as transcription factors. Since the DHHC protein domain
product of GRMZM2G068177, which was strongly suggested as a
candidate gene for the regulation of ear length, acts as an enzyme, this
may suggest a novel function of zinc finger proteins in monocot
reproductive organ development. However, further work is needed to
test this hypothesis. The third example, one QTL located on
chromosome 1 using K22/Dan340 RIL population which explains
11.8% of phenotypic variation for kernel number per row, also
overlapped with significant association signals (Figure 5G–I) Four
candidate genes: GRMZM2G088524, GRMZM2G022822, GR-
MZM2G108180 and GRMZM2G052666, located in a 200 kb
window around the significant signals, were predicted.
Discussion
The genome-wide imputation of genotypes has attracted much
attention given its broad applicability in the GWAS era. There are
a number of methods for imputing missing genotypes, but many
factors influence the accuracy of imputed genotypes [34,35]. In
this study, we proposed a two-step method combining IBD based
projection and KNN algorithm to infer missing genotypes,
resulting in 96.6% accuracy and 85.5% genome coverage in the
tested samples. Considering that the missing genotypes consist of
over 91.6% of our raw data set, this level of accuracy is acceptable.
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 Genome Wide Association Studies in Maize

Figure 4. Power comparisons in three simulation schemes for four different mapping methods: A-D, KW, MLM and LM. The ‘‘Power’’ was
defined as the detection frequency in 500 repeats for a certain QTN. For the purpose of computing power, a causal SNP was considered to be
detected only when the causal SNP was significant at a threshold from 1,000 times permutations. The power and type I error of major QTNs (A–C)

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and moderate QTNs (D–F) with common allele frequency. The power and type I error of major QTNs (G–I) and moderate QTNs (J–L) with rare allele
frequency. A-D test: Anderson–Darling test; LM: linear model; K-W test: Kruskal-Wallis test; MLM: mixed linear model. Scheme 1, phenotypes with
normal distribution; Scheme 2, phenotypes with abnormal distribution caused by uncertain effectors; Scheme 3, phenotypes with abnormal
distribution caused by a larger effect QTN with rare allele frequency.
doi:10.1371/journal.pgen.1004573.g004
Compared with other methods [4,17,34,35], the two-step method
has its advantages. Imputation based only on IBD regions ensures
high accuracy but a relatively low coverage rate. KNN algorithm
has been proven to be a good strategy for sequencing data [4],
however, it alone does not represent the true similarity of the
inbred lines due to low density of frame markers and rapid LD
decay in maize in our study. Therefore, we first used IBD based
imputation to increase marker density, and then the KNN
algorithm was used to infer the missing genotype, leading to high
coverage rate and imputation accuracy. Imputation error is often
caused by ignoring recombination and mutation within IBD
regions. In addition, if an inbred line with low density markers
share a region with two or more inbred lines with high density
markers and the missing genotypes are inferred on the basis of only
one of these lines, there is a high risk of error since the accuracy of
the projection depends on the identity between the projected and
chosen lines. Reanalysis of GWAS for kernel oil concentration
revealed consistent results and a higher detection power. Six more
associated loci were identified, most likely due to the increase in
sample size. The implication is that mapping resolutions are
enhanced by extracting moderately more information from the
genome and expanding sample size.
The detection of loci controlling complex traits using GWAS
has flourished and numerous statistical approaches for GWAS
analysis in plants have recently been described [14,36–40]. Linear
statistical models like ANOVA, general linear model (GLM), and
MLM establish significance cutoff by relying on the assumption
that target traits have normal distribution. However, sometimes
phenotype distribution in the moderate plant population is not
normal in the tails that may be due to the population size, field
experiment such as phenotyping errors, or genetic effects [31].
Based on our simulated data, nonparametric methods including A-
D and KW tests usually have greater power than LM and MLM
for abnormal phenotypes, rare alleles and moderate QTNs. It also
implies that A-D and KW tests should perform well to detect the
shifts of distribution as well as changes in the shape of distributions
[31]. A-D test possesses advantages than K-W test in the detection
of QTNs with rare alleles. However, MLM performs better than
A-D and KW test for the major QTNs especially those with
common alleles. However, we need to keep in mind that
population structure of the studied samples is the key confounder
for GWAS. In the measured 17 agronomic traits of present study,
we observed the phenotypic variation explained by population
structure ranged between 0.9% and 32.3% (Table S2). In the A-D
test, we didn’t account the confounding by population structure in
the subpopulation that may lead to false-positive findings.
Genomic control is a good alternative for controlling the statistic
inflations [27–30], different inflation factors were observed in
different traits and different subpopulations in present study (Table
S7). We detected 19 loci significantly associated with 13 traits at
the Bonferroni-corrected threshold (2log P.5.74, a = 1) (Table
S7) using genomic-control (lregress ) to adjust our real phenotype
test statistic from A-D test. However, we also need to be careful
that the adjusted 2Log P might be over corrected, since A-D test
has already controlled part of the population structure and
genomic control method is affected significantly by the true
association signals , even for the agronomic traits may involve a
larger number of loci with small effects [21,36,37,41]. And the
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Genome Wide Association Studies in Maize
influence of epistatic genetic effect to the genomic control is still
not explored [27–30]. Another thing need to be noticed is testing
within subpopulations (A-D test) and across the whole panel with
controlling the population structure (MLM) are different. Testing
within subpopulations changes allele frequencies of background
alleles and therefore possibly changes the epistatic interactions that
are mapped in an additive manner within subpopulations but were
not mapped across populations.
In general, A-D test could be a good complement to current
popular GWAS methods. As each method owning its own
advantages, the preliminary understanding of the traits studied is
needed for choosing GWAS methods or trying different GWAS
methods would be helpful especially for those studies only few or
none significant signals were identified by using only one method.
In this study, we performed GWAS using both MLM and A-D test
for 17 agronomic traits. In total, 18 overlapped regions were
detected by the two approaches (Table S7). The A-D test also
showed high concordance with previous studies in identifying a
higher number of QTL related to agronomic traits.
Our noticed nonparametric statistical approach is robust with
respect to non-normality, similarly to the KS test [12]. The KS test
tends to be more sensitive around the median value and less
sensitive at the extreme ends of the distribution. Thus, the KS test
is not always appropriate for calculating the significance of data
sets which differs at the tails of the probability distribution, while
the median remains unchanged [31]. The A-D test improves upon
the KS test because it has more sensitivity towards the tails of the
pooled sample. More importantly, the performance of the A-D test
for small samples is quite good, as demonstrated by numerous
Monte Carlo simulations [19]. This means that, for complex traits,
the A-D test can make a good use of SNPs that have minor allele
frequency and keep detection ability to the relatively small effect
loci. At the same time, it is important to recognize that there are
always limitations to what can be achieved using statistics. It seems
that A-D test does not work well for all traits. Interestingly, we
identified 14.6% associations by A-D test that differed in the width
of the phenotype distribution but which had nearly identical trait
means (Figure 3A). In these cases the differences between
distributions are real, but the corresponding genetic markers
would not be useful in breeding if the objective is to change the
phenotypic means. Instead, the associations appear to represent
allelic differences in the apparent trait stability. Therefore, to
confirm candidate loci, it is necessary to check both frequency
distribution and normality of the distribution curves (Figure 3).
Several studies in humans have confirmed that using multiple
methods for statistical inference critically enables the interpreta-
tion of results and engenders stronger candidates for experimental
follow-up [42].
We identified some genes affecting important agronomic traits
in maize that are very good candidates for future detailed analysis,
for allele mining to identify functional variation, and for marker
development. As whole genome sequences become available for
many crop species including maize, as well as for multiple
genotypes of the same species through resequencing, along with
cost-effective high-throughput genotyping systems and the next
generation of sequencing technologies, GWAS becomes practical
and its use in plant breeding will allow the manipulation of many
traits at the whole-genome level. Association mapping using a set
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Figure 5. Co-localization of association peaks, QTL and well-
annotated candidate genes. A. Significant association signals on
chromosome 7 for kernel width; B. A major kernel width QTL
(R2 = 18.7%) was mapped on chromosome 7 from 129 Mb to 149 Mb
with BK/Yu8701 RILs and covered the significant association signals; C.
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Genome Wide Association Studies in Maize
The phenotype’s frequency distribution histogram and normal distri-
bution curve at the peak SNP of kernel width; D. Significant association
signals on chromosome 2 for ear length; E. A major ear length QTL
(R2 = 5.7%) was mapped on chromosome 2 in B73/By804 RILs and
covered the significant association signals; F. The phenotype’s
frequency distribution histogram and normal distribution curve at the
peak SNP of ear length; G. Significant association signals on chr1 for
kernel number per row; H. A major kernel number per row QTL
(R2 = 11.78%) was mapped on chr1 in K22/DAN340 RILs and covered the
significant association signals; I. The frequency distribution histogram
and normal distribution curve at the peak SNP of kernel number per
row.
doi:10.1371/journal.pgen.1004573.g005
of global diverse breeding germplasm and high-throughput SNP
markers, as shown in this study, provides high-resolution dissection
of the genetic architecture of complex traits. This knowledge in
turn will be useful not only for designing marker-assisted selection
strategies but also for optimizing conventional breeding systems.
Materials and Methods
Plant material and phenotyping
A total of 513 maize lines with tropical, subtropical and
temperate backgrounds representing the global maize diversity
were employed for genome-wide association mapping in this
study. All maize inbred lines have been well described in previous
studies [13,15] and the 513 maize lines were classified into four
subgroups based on population structure Q matrix: Stiff stalk (SS)
with 112 lines, Non-stiff stalk (NSS) with 116 lines, Tropical-
subtropical (TST) with 258 lines, and an admixed group with 27
lines (detailed information also can be downloaded at (www.
maizego.org/resource). A Randomized complete block design
with one to two replications was used for field trials in five
environments, including Ya’an (30uN, 103uE), Sanya (18uN,
109uE), Yunnan (25uN, 102uE) in 2009, Guangxi (23uN, 110uE)
and Yunnan (25uN, 102uE) in 2010. A row length of 3 m was used
for each line including 11 plants plot21 with 25 cm plant to plant
and 60 cm row to row distance. Five randomly selected plants
were used for phenotypic data acquisition in each line and the
mean data in each replication was used for phenotypic analysis. A
total of 17 economically important traits were phenotyped (Table
S1). These traits were divided into three categories: morphological
attributes (plant height, ear height, ear leaf width and length, tassel
main axis length, tassel branch number, and leaf number above
ear), yield related traits (ear length and diameter, cob diameter,
kernel number per row, 100-grain weight, cob weight and kernel
width), and maturity traits (days to heading, anthesis, and silking).
Best linear unbiased predictions (BLUP) for each line across five
environments were calculated using the MIXED procedure in
SAS (Release 9.1.3; SAS Institute, Cary, NC), and employed for
evaluating trait variation in the association panel.
Imputation yield and accuracy
Imputation methods have not been developed to deal specifi-
cally with low density of SNP marker data. Of the available
imputation models, identity by descent (IBD) based projection [17]
and the k-nearest neighbor algorithm (KNN) [4] seemed to
effectively infer a large number of missing genotypes. To assess the
performance of IBD based projection, preliminary tests for
chromosome 1 in 368 maize lines were conducted. We removed
genotype data without frame SNPs and then compared the
observed genotypes with those generated by projection. The
number of IBD regions with consecutive SNPs for 368 lines varied
from 1 to 285 on chromosome 1, and projection accuracy, defined
as the percentage of correctly projected genotypes ranged from
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74.8% to 99.1%, with an average of 92.6% (Table S3). The mean
error ratio pooled over in 32,015 IBD regions on chromosome 1
for the 368 maize lines was also calculated (Figure S19A, B), and
gradually declined with the increasing number of identical SNP
and size in IBD regions. Preliminary testing suggested that IBD
regions with 150 consecutive SNPs and a size of 5 Mb or more
were highly conserved in maize and error rate for projection was
well controlled within 5% (Figure S19A, B). The number of
qualified IBD segments ranged from 0 to 20 for 368 lines and
coverage rate, defined as the percentage of projected genotypes,
accounted for 61.99% of genomic regions on chromosome 1, with
projection accuracy increased from 92.59% to 96.62% on average
(Table S3). Therefore, IBD based projection for regions with 150
consecutive SNPs and a size of 5 Mb were applied for integration
of SNPs from RNA-seq data set onto the 145 additional maize
inbred lines. Alternatively, the K-Nearest Neighbor (KNN)
algorithm was also used to enrich the physical map of each line
constructed by 56,110 SNPs from MaizeSNP50 chip by inferring
the missing genotypes of the unique loci from RNA-seq SNP data.
In the preliminary test, this method was efficient and the
imputation accuracy and coverage rate for 368 lines were
97.48% and 75.35%, respectively (Table S3). The IBD based
projection and KNN imputation revealed high inferred accuracy;
however, the coverage rates were relatively low, with an average of
62% and 75%, respectively. In order to increase the coverage rate
and keep high imputation accuracy, IBD based projection and
KNN algorithm were combined to infer missing genotypes. The
IBD method can provide more frame SNPs for the KNN
algorithm, and simultaneously the KNN algorithm compensates
for the weakness of the IBD method in coverage rate. About 38%
of the genotypes were missing after prediction of IBD regions with
150 consecutive SNPs and 5 Mb size, and then the KNN
algorithm was used to impute the missing data, resulting in
95.8% of accuracy for the missing data. The joint IBD based
projection and KNN imputation of the genotypes of 368 lines
increased coverage rate from 62% to 87.2%, with a total accuracy
of 95.9% in the preliminary test. The projection accuracy was also
affected by heterozygosity of each line, which increased from
95.88% to 96.60% after excluding 44 lines with more than 10%
heterozygosity. The joint IBD based projection and KNN
imputation that performed well in the preliminary test was used
for the integration of SNPs from the high density SNPs data set
onto 145 maize lines genotyped by 56110 SNPs. For 145 maize
lines, 54.18% and 32.28% of loci across 10 chromosomes were
inferred through IBD based projection and subsequent KNN
imputation, respectively. As a result, 85.46% of loci for the whole
maize genome were filled. The average density for the whole panel
increased from 20 SNPs to more than 200 SNPs per Mb.
QTL mapping
The linkage analyses of ear length, kernel number per row, and
kernel width were performed in three recombination inbred line
(RIL) populations, BY804/B73 (197 individuals), K22/Dan340
(197 individuals), and BK/Yu8701 (165 individuals). All the RIL
lines and their parents were genotyped using Maize SNP50 assays
(Illumina) containing 56,110 SNPs [14]. The phenotype of BK/
Yu8701 in Henan 2011 and BLUP value from 5 environments of
BY804/B73 and K22/Dan340 were used. QTL mapping using
the composite interval mapping method [43] was performed in the
package QTL cartographer version 2.5 [44].
Statistical analysis and association mapping
ANOVA, correlation, and repeatability analyses for 17 agro-
nomic traits were conducted using SAS software (Release 9.1.3;
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Genome Wide Association Studies in Maize
SAS Institute, Cary, NC). Heritability analysis and association
analysis for the 17 agronomic traits in Data set 2 were conducted
by MLM using TASSEL [45] software package. The observed p
values from marker-trait associations were used to display Q-Q
plots and Manhattan plots, using R. Permutation tests were used
to determine the cutoff for GWAS. Considering the computation
time, we only choose three typical traits with different population
structure effects (kernel width, ear height and day of flowering
time) as examples. The results showed that the cutoff values are
similar with the Bonferroni correction. To simplify the procedures,
we use the uniform Bonferroni-corrected thresholds at a = 1 and
a = 0.05 as the cutoffs. When performing n tests, if the significance
level for the entire series of tests is a, then each of the tests should
have a probability of P = a/n. When the numbers of markers was
556809 SNPs, at a = 1 and a = 0.05, the Bonferroni-corrected
thresholds for the p values were 1.79661026 and 8.9561028, with
corresponding 2log p values of 5.74 and 7.05, respectively.
Regression estimator (lregress ) of Genomic Control inflation factor
was used [28]. Percentage of PVE by associated SNPs was
calculated by ANOVA. Informative SNPs and candidate genes at
the identified loci for the corresponding traits were from public
maize genome data set B73 RefGen_v2.
Simulation study
To compare the power and FDR of A-D test, Kruskal-Wallis
(K-W test) test, linear model (LM) and mixed linear model (MLM),
three schemes with different phenotype distribution were simulat-
ed by considering the QTN effects and allele frequency.
Scheme 1 was used to simulate a normal distribution phenotype
with the contribution of population structure, additive genetic
effect, epistatic genetic effect and residual effect [6]. The
population structure and epistasis explained 10% of the total
phenotypic variation, respectively. The additive effect was the sum
of all additive effects for 20 causal QTNs. For approaching the real
genetic architecture, we set 20% major QTNs explaining 30% of
the sum of all assigned genetic effect and 80% moderate QTNs
explaining 70% of the sum of all assigned genetic effect. Half of
major and moderate QTNs were rare alleles (MAF = 0.05–0.1)
and half were common alleles (MAF = 0.25–0.45). Larger genetic
effects were assigned to the rare alleles QTNs to ensure them could
explain the same proportion of phenotypic variation as common
alleles QTNs. The ratio of assigned genetic effects between rare
alleles QTNs (at MAF = 0.075) and common alleles QTNs (at
MAF = 0.35) was calculated based on 1=(1z1=p(1{p)k2 ). The
genetic effect was assigned to all SNPs, one at a time [6]. The
proportion of the additive effect was defined by narrow-sense
heritability which is the proportion of additive variance over the
total variance, and h2 ~0:7 was examined. The residual effect
followed a normal distribution and had a variance to satisfy the
contributions from additive and epistatic effects at the designated
level [6].
Scheme 2 was used to simulate an abnormal distribution
phenotype with a long tail on one side. On the basis of scheme 1,
10% of lines were randomly selected and added an extra residual
effect (1 to 6 fold standard deviation of the phenotype). All the
others were same.
Scheme 3 was designed to simulate an abnormal distribution
phenotype caused by a larger effect background rare QTN. The
additive effect was still the sum of all additive effects for 20 causal
QTNs. 1 background QTN, 3 major QTNs and 16 moderate
QTNs explaining 25%, 20%, 60% of the sum of all assigned
genetic effect respectively. The population structure effect,
epistatic effect and residual effect were consistent with scheme 1.
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 Genome Wide Association Studies in Maize

Simulations of the phenotypes were repeated 500 times in all where:

schemes. All simulated phenotypes had been analyzed with the
four methods presented in the main text. 1,000 permutations had Xk 1 XN{1 1 XN{2 XN{1 1
been done separately for the four methods to obtain the threshold S~ , T~ , g~
i~1 ni i~1 i i~1 j~iz1 (N{i)j
at different type I error risk.

Anderson-Darling test 3. Calculate TkN :

The Anderson-Darling two-sample procedure assumes that the
two samples have a continuous distribution function and we are A2kN {(k{1)
interested in testing the null hypothesis that the two phenotype TkN ~
sN
samples divided by two alleles of one SNP have the same
distribution, without specifying the nature of population:
H0 : F1 ~F2 4. Refer TkN to the upper a percentiles tm (L) of the Tm
The test procedure is as follows: distribution table below, reject H0 at significance level a if TkN
1. Calculate A2kN : exceeds the given point tk{1 (a).If TkN is outside the range of the
The computational formula for A2kN not adjusted for ties is, table. Plotting the log-odds of a versus t1 (a), a strong linear pattern
indicates that simple linear extrapolation should give good
approximate p values.
1 Xk 1 XL{1 (nFij {jni )2
A2kN ~ ½ 
N i~1 n
i
j~1 j(N{j)
L 0:25 0:10 0:05 0:025 0:01
and the corresponding adjusted for ties is, t1 ðLÞ 0:326 1:225 1:960 2:719 3:752

where:
N{1 Xk 1 XL (NFij {ni Hj )2
A2kN ~ 2
½ 
N i~1 ni j~1 Hj (N{Hj ){Nhj =4
m~k{1
where:
F1 , F2 indicates the two phenotype distribution function
URL. One R package (ADGWAS) for GWAS by Anderson-
k = 2; i = 1, 2
Darling test can be downloaded here: http://www.maizego.org/
ni = data number in the ith sample; j = 1,2,…,ni
Resources.html
N = total number of two samples’ individuals; N~n1 zn2
xij = data in the i sample and j observation within that sample
Supporting Information
L = the number of unique data, where it will be less than n with
tied data Figure S1 Genome-wide association analysis of plant height. (A,
z(j) = distinct values of all combined data ordered in ascendant B) Phenotype histogram and distribution of subpopulations in 513
way denoted z(1),z(2),…,z(L) maize lines. (C) Manhattan plots of mixed linear model conducted
hj = number of values in the pooled sample equal to z(j) in imputation data, respectively. (D) Quantile-Quantile plots of p-
Hj = number of values in the combined samples less than z(j) values of mixed linear model conducted in imputation data. Know
plus one half of the number of values in the combined samples genes controlling the traits were labeled. (E) Summary of GWAS
equal to z(j) results from Anderson-Darling test performed on each subpopu-
Fij = number of values in the ith sample which are small than lation independently for plant height. The three subpopulations:
z(j) plus one half the number of values in this sample which are SS (subpop-1), NSS (subpop-2) and TST (subpop-3).
equal to z(j) (TIF)
2. Calculate sN : Figure S2 Genome-wide association analysis of ear height. (A,
Under H0 , the variance of A2kN is, B) Phenotype histogram and distribution of subpopulations in 513
maize lines. (C) Manhattan plots of mixed linear model conducted
in imputation data, respectively. (D) Quantile-Quantile plots of p-
aN 3 zbN 2 zcNzd
s2N ~var(A2kN )~ values of mixed linear model conducted in imputation data. (E)
(N{1)(N{2)(N{3) Summary of GWAS results from Anderson-Darling test performed
on each subpopulation independently for ear height.
with:
(TIF)
Figure S3 Genome-wide association analysis of ear leaf width.
a~(4g{6)(k{1)z(10{6g)S
(A, B) Phenotype histogram and distribution of subpopulations in
513 maize lines. (C) Manhattan plots of mixed linear model
conducted in imputation data, respectively. (D) Quantile-Quantile
b~(2g{4)k2 z8Tkz(2g{14h{4)S{8Tz4g{6 plots of p-values of mixed linear model conducted in imputation
data. (E) Summary of GWAS results from Anderson-Darling test
performed on each subpopulation independently for plant ear leaf
c~(6Tz2g{2)k2 z(4T{4gz6)kz(2h{6)Sz4T width.
(TIF)
Figure S4 Genome-wide association analysis of ear leaf length.
d~(2Tz6)k2 {4Tk (A, B) Phenotype histogram and distribution of subpopulations in

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513 maize lines. (C) Manhattan plots of mixed linear model
conducted in imputation data, respectively. (D) Quantile-Quantile
plots of p-values of mixed linear model conducted in imputation
data. (E) Summary of GWAS results from Anderson-Darling test
performed on each subpopulation independently for ear leaf
length.
(TIF)
Figure S5 Genome-wide association analysis of tassel main axis
length. (A, B) Phenotype histogram and distribution of subpop-
ulations in 513 maize lines. (C) Manhattan plots of mixed linear
model conducted in imputation data, respectively. (D) Quantile-
Quantile plots of p-values of mixed linear model conducted in
imputation data. Know genes controlling the traits were labeled.
(E) Summary of GWAS results from Anderson-Darling test
performed on each subpopulation independently for tassel main
axis length.
(TIF)
Figure S6 Genome-wide association analysis of tassel branch
number. (A, B) Phenotype histogram and distribution of
subpopulations in 513 maize lines. (C) Manhattan plots of mixed
linear model conducted in imputation data, respectively. (D)
Quantile-Quantile plots of p-values of mixed linear model
conducted in imputation data. Know genes controlling the traits
were labeled. (E) Summary of GWAS results from Anderson-
Darling test performed on each subpopulation independently for
tassel branch number.
(TIF)
Figure S7 Genome-wide association analysis of leaf number
above ear. (A, B) Phenotype histogram and distribution of
subpopulations in 513 maize lines. (C) Manhattan plots of mixed
linear model conducted in imputation data, respectively. (D)
Quantile-Quantile plots of p-values of mixed linear model
conducted in imputation data. (E) Summary of GWAS results
from Anderson-Darling test performed on each subpopulation
independently for leaf number above ear.
(TIF)
Figure S8 Genome-wide association analysis of ear length. (A,
B) Phenotype histogram and distribution of subpopulations in 513
maize lines. (C) Manhattan plots of mixed linear model conducted
in imputation data, respectively. (D) Quantile-Quantile plots of p-
values of mixed linear model conducted in imputation data. Know
genes controlling the traits were labeled. (E) Summary of GWAS
results from Anderson-Darling test performed on each subpopu-
lation independently for ear length.
(TIF)
Figure S9 Genome-wide association analysis of ear diameter.
(A, B) Phenotype histogram and distribution of subpopulations in
513 maize lines. (C) Manhattan plots of mixed linear model
conducted in imputation data, respectively. (D) Quantile-Quantile
plots of p-values of mixed linear model conducted in imputation
data. Know genes controlling the traits were labeled. (E) Summary
of GWAS results from Anderson-Darling test performed on each
subpopulation independently for ear diameter.
(TIF)
Figure S10 Genome-wide association analysis of cob diameter.
(A, B) Phenotype histogram and distribution of subpopulations in
513 maize lines. (C) Manhattan plots of mixed linear model
conducted in imputation data, respectively. (D) Quantile-Quantile
plots of p-values of mixed linear model conducted in imputation
data. Know genes controlling the traits were labeled. (E) Summary
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Genome Wide Association Studies in Maize
of GWAS results from Anderson-Darling test performed on each
subpopulation independently for cob diameter.
(TIF)
Figure S11 Genome-wide association analysis of kernel number
per row. (A, B) Phenotype histogram and distribution of
subpopulations in 513 maize lines. (C) Manhattan plots of mixed
linear model conducted in imputation data, respectively. (D)
Quantile-Quantile plots of p-values of mixed linear model
conducted in imputation data. Know genes controlling the traits
were labeled. (E) Summary of GWAS results from Anderson-
Darling test performed on each subpopulation independently for
kernel number per row.
(TIF)
Figure S12 Genome-wide association analysis of 100-grain
weight. (A, B) Phenotype histogram and distribution of subpop-
ulations in 513 maize lines. (C) Manhattan plots of mixed linear
model conducted in imputation data, respectively. (D) Quantile-
Quantile plots of p-values of mixed linear model conducted in
imputation data. Know genes controlling the traits were labeled.
(E) Summary of GWAS results from Anderson-Darling test
performed on each subpopulation independently for 100-grain
weight.
(TIF)
Figure S13 Genome-wide association analysis of cob weight. (A,
B) Phenotype histogram and distribution of subpopulations in 513
maize lines. (C) Manhattan plots of mixed linear model conducted
in imputation data, respectively. (D) Quantile-Quantile plots of p-
values of mixed linear model conducted in imputation data. Know
genes controlling the traits were labeled. (E) Summary of GWAS
results from Anderson-Darling test performed on each subpopu-
lation independently for cob weight.
(TIF)
Figure S14 Genome-wide association analysis of kernel width.
(A, B) Phenotype histogram and distribution of subpopulations in
513 maize lines. (C) Manhattan plots of mixed linear model
conducted in imputation data, respectively. (D) Quantile-Quantile
plots of p-values of mixed linear model conducted in imputation
data. (E) Summary of GWAS results from Anderson-Darling test
performed on each subpopulation independently for kernel width.
(TIF)
Figure S15 Genome-wide association analysis of days to
anthesis. (A, B) Phenotype histogram and distribution of
subpopulations in 513 maize lines. (C) Manhattan plots of mixed
linear model conducted in imputation data, respectively. (D)
Quantile-Quantile plots of p-values of mixed linear model
conducted in imputation data. Know genes controlling the traits
were labeled. (E) Summary of GWAS results from Anderson-
Darling test performed on each subpopulation independently for
days to anthesis.
(TIF)
Figure S16 Genome-wide association analysis of days to silking.
(A, B) Phenotype histogram and distribution of subpopulations in
513 maize lines. (C) Manhattan plots of mixed linear model
conducted in imputation data, respectively. (D) Quantile-Quantile
plots of p-values of mixed linear model conducted in imputation
data. (E) Summary of GWAS results from Anderson-Darling test
performed on each subpopulation independently for days to
silking.
(TIF)
Figure S17 Genome-wide association analysis of days to
heading. (A, B) Phenotype histogram and distribution of
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subpopulations in 513 maize lines. (C) Manhattan plots of mixed
linear model conducted in imputation data, respectively. (D)
Quantile-Quantile plots of p-values of mixed linear model
conducted in imputation data. Know genes controlling the traits
were labeled. (E) Summary of GWAS results from Anderson-
Darling test performed on each subpopulation independently for
days to heading.
(TIF)
Figure S18 Pair-wise Pearson’s correlation among 17 traits in
513 maize lines.
(TIF)
Figure S19 The mean error ratio and mean coverage ratio pool
over SNP number within IBD region (A) and size of IBD region
(B), respectively, on chromosome 1 for the 368 maize lines. (C)
Imputation accuracy and filling rate for each of 72 combinations
of variables of KNN. The combination, indicated by arrow, was
chosen for final data imputation.
(TIF)
Table S1 Description of the traits evaluated in the study.
(XLSX)
Table S2 Phenotype variation of 17 agronomic traits in 513
maize lines. a ANOVA, analysis of variance, showing the mean
square and degrees of freedom (in parentheses). The F-test was
applied to determine the significance level. Both the environments
and lines were fitted in the model as random effects. ** indicate
significance at level of 0.001; s.d., standard deviation. bPVE by Q,
the percentage of phenotypic variance explained by the subpop-
ulation structure.
(XLSX)
Table S3 IBD base projection and KNN imputation for
validation dataset.
(XLSX)
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We gratefully thank the four anonymous reviewers for their valuable
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